Maize plants can enter a standby mode to cope with chilling stress

European Flint maize inbred lines are used as a source of adaptation to cold in most breeding programs in Northern Europe. A deep understanding of their adaptation strategy could thus provide valuable clues for further improvement, which is required in the current context of climate change.

R E S E A R C H A R T I C L E Open Access

Maize plants can enter a standby mode

to cope with chilling stress

Lặtitia Riva-Roveda1,2,4, Brigitte Escale1, Catherine Giauffret2,3and Claire Périlleux4*

Abstract

Background: European Flint maize inbred lines are used as a source of adaptation to cold in most breeding

programs in Northern Europe A deep understanding of their adaptation strategy could thus provide valuable clues for further improvement, which is required in the current context of climate change We therefore compared six inbreds and two derived Flint x Dent hybrids for their response to one-week at low temperature (10 °C day/7 or 4 °C night) during steady-state vegetative growth

Results: Leaf growth was arrested during chilling treatment but recovered fast upon return to warm temperature, so that no negative effect on shoot biomass was measured Gene expression analyses of the emerging leaf in the hybrids suggest that plants maintained a‘ready-to-grow’ state during chilling since cell cycle genes were not differentially expressed in the division zone and genes coding for expansins were on the opposite up-regulated in the elongation zone In photosynthetic tissues, a strong reduction in PSII efficiency was measured Chilling repressed chlorophyll biosynthesis; we detected accumulation of the precursor geranylgeranyl chlorophyll a and down-regulation of GERANYLGERANYL REDUCTASE (GGR) in mature leaf tissues Excess light energy was mostly dissipated through fluorescence and constitutive thermal dissipation processes, rather than by light-regulated thermal dissipation Consistently, only weak clues of xanthophyll cycle activation were found CO2assimilation was reduced by

chilling, as well as the expression levels of genes encoding phosphoenolpyruvate carboxylase (PEPC), pyruvate orthophosphate dikinase (PPDK), and the small subunit of Rubisco Accumulation of sugars was correlated with a strong decrease of the specific leaf area (SLA)

Conclusions: Altogether, our study reveals good tolerance of the photosynthetic machinery of Northern European maize to chilling and suggests that growth arrest might be their strategy for fast recovery after a mild stress

Keywords: Maize (Zea mays), Cold tolerance, Leaf growth, Photoprotection

Background

Tolerance to cold has been a long lasting issue for maize

cultivation Extension from its native tropical area in

Southwestern Mexico toward Northern countries indeed

required selection of short-cycle varieties to alleviate the

prolongation of growth duration by low temperature

Prominent in maize history is the early flowering Northern

Flint race that adapted to cold temperate regions of

Northeastern America and was introduced in Northern

Europe probably at the beginning of the 16th century [1]

Interestingly, in both American and European continents,

Northern races were hybridized with late materials to pro-duce new types adapted to mid-latitude climates, such as Corn Belt Dent in America resulting from the inter-crossing between Northern Flint and Southern Dent races [2] After World War II, traditional landraces were pro-gressively replaced by hybrid varieties [3] European Flint inbred lines provided valuable traits for regions with cool and wet spring conditions: cold tolerance, early vigor and short growing cycles [4] Thanks to their good heterotic pattern with American Dent material, they have been widely used in Northern Europe for hybrid production [5] New challenges give to cold tolerance of maize a renewed interest The current climate change encour-ages early planting that potentially increases yield and participates to water deficit avoidance in summer Early harvesting is also suitable to prevent fungal growth and

* Correspondence: cperilleux@ulg.ac.be

4 InBioS, PhytoSYSTEMS, Laboratory of Plant Physiology, University of Liège,

Sart Tilman Campus Quartier Vallée 1, Chemin de la Vallée 4, B-4000 Liège,

Belgium

Full list of author information is available at the end of the article

© 2016 The Author(s) Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver

mycotoxin production in grain and to reduce drying

costs In France, for example, the mean sowing date has

advanced from 5 to 15 days over the past 30 years and

this trend will probably increase in the future [6]

How-ever, earlier sowing dates increase the risk of exposure of

the plants to cold and hence require to re-evaluate

culti-vated materials and to select inbreds that are more

toler-ant to low temperatures

Assessing cold tolerance in maize requires the choice

of an experimental design The conditions of stress

oc-currence, its intensity and duration as well as the

devel-opmental stage of the plants, are all critical parameters

that delimit the scope of the research On the one hand,

laboratory experiments were, and still are, instrumental

in identifying the physiological and cellular effects of

cold They are generally performed on young seedlings

of reference genotypes, often inbred lines such as B73,

transferred into cold rooms (<5 °C) for a short period

This kind of approach clearly demonstrated that cold

impairs photosynthetic machinery and unveiled the

physiological mechanisms of cold tolerance [7, 8] On

the other hand, field experiments explore genetic

diver-sity within large sets of genotypes to reliably estimate

the relationship between cold tolerance traits and

agro-nomic performance Growth of maize is strongly limited

below 15 °C [9] and ‘chilling’ commonly refers to the

temperature range between 5 and 15 °C [8] Reduced leaf

growth at these temperatures implies that light

intercep-tion area is limited and, in addiintercep-tion to impaired

photo-synthesis, contributes to depressed plant productivity in

terms of biomass or grain yield [10] Numerous

quanti-tative traits (QTL) have been identified but were only

partially consistent across the different mapping

popula-tions used, indicating a strong influence of the genetic

background [11] Moreover, assessment of chilling

toler-ance in the field is hampered by the fact that fluctuating

environments and occurrence of multiple stresses

com-plicate the identification of causal relationships between

chilling and final yield Several authors have reported

poor correlations between widely used traits, e.g., vigor

of seedlings is neither positively associated with grain

yield [12] nor with dry matter accumulation [13]

Select-able phenotypic traits thus remain an issue for breeding

cold tolerance in maize

European Flint inbred lines are still used as a source of

adaptation to cold in most maize breeding programs in

Northern Europe [3] and hence a deep understanding of

their adaptation strategy could provide valuable clues for

further improvement The effects of cold on cellular

pro-cesses governing leaf growth and photosynthesis can be

assessed simultaneously along the maize leaf which

shows a longitudinal gradient with proliferative,

expand-ing and mature cells located at increasexpand-ing distance from

the base New markers could be discovered thanks to

the availability of transcriptome profiles associated with this developmental gradient [14] and from homology to known regulatory networks disclosed in Arabidopsis, e.g., cell cycle genes [15] or cold-signalling components [7, 16] The best understood cold response pathway in plants involves CBF (C-repeat/drought-responsive elem-ent binding) transcription factors, also known as DREB1, which activate target genes [17] ICE1 (inducer of CBF/ DREB expression 1) is an upstream regulator of the ex-pression of theCBF/DREB1 genes and is itself activated

by cold at the post-translational level [18]

The growth gradient of the maize leaf is established early after initiation and persists after the proliferative zone splits to occupy a region at the base of the blade and at the base of the sheath, separated by the ligule [19] Chilling can then restrict growth of the blade and/

or the sheath of the leaves, by impairing cell prolifera-tion and/or cell expansion, depending on their develop-mental stage [15, 20] In addition, when chilling occurs soon after leaf initiation, the reduction of cell division rate can be partly compensated by an increase in cell length [21] Cold compromises the assembly of the photosynthetic apparatus in differentiating cells [22], or its efficiency and integrity in mature tissues [23, 24] Cold reduces the velocity of enzymatic reactions such as those catalysed, in C4 species like maize, by phospho-enolpyruvate carboxylase (PEPC), pyruvate orthophos-phate dikinase (PPDK), or Rubisco [25] The resulting decrease in CO2assimilation leads to saturation of the electron transport [26] Light energy that is absorbed in excess can lead to photooxidation and damage to mem-brane proteins unless it is re-emitted as chlorophyll fluorescence or dissipated as heat Changes in fluores-cence yield thus reflect changes in photochemical (PSII efficiency, ΦPSII) and non-photochemical quenching (NPQ) of excitation energy and are widely used as physiological proxy of cold tolerance [26] Heat dissipa-tion is stimulated by the acidificadissipa-tion of the thylakoid lumen, which activates psbS, a PSII protein embedded

in the thylakoid membrane, and the xanthophyll cycle, i.e., the reversible de-epoxidation of violaxanthin into dissipative zeaxanthin In case these photoprotective mechanisms are not sufficient, PSII reaction centers are subject to permanent damage, a process called photoin-hibition (reviewed in [27])

The aim of this study was to characterize maize in-breds adapted to temperate climate for their tolerance to chilling Since prediction of hybrid tolerance from parental inbreds may not be reliable [28], we also analysed derived Flint x Dent hybrids As we expected subtle differences for cold tolerance in this material, the experiments were per-formed in controlled growth chambers, which provide an appropriate scale for dissecting the mechanisms of chilling tolerance in reproducible conditions [29] Since chilling at

early stages was often studied in the literature and

ap-peared to have few consequences on later development

[13, 21], we focused on the steady-state vegetative

growth of the plants We used an array of physiological,

biochemical and molecular parameters in order to gain

an overview of the adaptive mechanisms that could

ac-count for cold tolerance and help to define selectable

phenotypic traits

Methods

Plant material and culture conditions

Three unrelated Flint lines (F2, F283, F03802) released

in 1958, 1985 and 2008, respectively, and three Dent

lines (F353, B73, Mo17) representing Iodent/European

Dent, Stiff Stalk and Lancaster heterotic groups,

re-spectively, were provided by INRA Two hybrids

(F03802xF353, F2xF353) were produced during the

growing season 2011–2012 in Graneros (Chile) and

during summer 2013 in Montardon (France)

Grains were sown in compressed peat pots (Jiffy pots,

Ets Lejeune, Warsage, Belgium) filled with horticultural

compost (TYPical Tonerde I, Brill substrate GmbH &

Co., Georgsdorf, Germany; www.brill-substrate.com)

When the third leaf emerged, seedlings were

trans-planted into larger pots (14-cm diameter, 10-cm height)

filled with the same compost supplemented with

10 % perlite (v/v) A nylon mesh was inserted in the

bottom of the pot and was soaked in a reservoir to

provide complete nutrient solution continuously (12.6

mEquiv N l−1) Plants were grown in controlled

cabi-nets (Conviron, Winnipeg, Canada) in 16-h long days,

300 μmol m−2 s−1 (inbred lines) or 400 μmol m−2 s−1

(hybrids) of photosynthetically active radiation (PAR)

being provided at the canopy level by 54-W

fluores-cent tubes and 40-W incandesfluores-cent bulbs (ratio 3:1)

Air relative humidity was 70 %

Temperature treatments for inbreds and hybrids

Standard temperature was 24 °C day/18 °C night For

cold treatment, plants were transferred to 10 °C day/7 °C

night (inbred lines) or 10 °C day/4 °C night (hybrids) for

one week

Experimental series comprised 15 plants of each

geno-type, in control and treated (cold) conditions The

num-bers of plants and replications used for all traits

measured are detailed in the table and figure legends

Growth measurements

The visible leaf stage (VL) was defined as the total

num-ber of visible leaves counted with the plant held at eye

level For the youngest emerging leaf, the decimal stage

was evaluated as in [30]: the ratio (V/T) between the

length of the visible portion (V) and the total length of

the leaf measured from its tip to the base of the plant

(T) was divided by its maximum value (V/T)maxreached

at the time the next leaf emerged In practice, (V/T)max

was between 0.47 and 0.60 for the genotypes used in this study The phyllochron is the time (days) between suc-cessive leaf appearance and was calculated as the inverse

of mean VL increase rate

Leaf growth was estimated by leaf elongation rate and final leaf length Leaf elongation rate was estimated by measuring the elongation of the visible part of the leaf until it reached its final length In order to avoid cumu-lating leaf and stem growth in leaf elongation rate calcu-lation, length was measured from the tip of the growing leaf to the ligule of the youngest fully expanded leaf below it End-point measurement of leaf length was made at 12-VL stage Specific Leaf Area (SLA, cm2g dry weight−1) determination was made on 8-mm diameter leaf discs harvested at mid-length of leaf 4, outside the mid-vein, at the end of the cold treatment

Photosynthetic parameters measurements Photosynthetic performance was evaluated by measur-ing the maximum quantum yield of photosystem II (Fv/ Fm) and quantum efficiencies of photochemistry (ФPSII = 1 – Fs/Fm’), of regulated thermal energy dissi-pation (ФNPQ = Fs/Fm’ – Fs/Fm) and of constitutive thermal and radiative (fluorescence) energy dissipation (Φf,D = Fs/Fm) [31] The sum of the three yields (ΦPSII, ΦNPQ and Φf,D) is equal to 1 and reflects light energy partitioning In practice, we measured fluores-cence at mid-length of leaf 4 emerged part after adapta-tion in darkness (at least 20 min) or in the light (at least 6 min at 300 or 400 μmol m−2 s−1 PAR) using a Handy PEA fluorimeter (Hansatech) The measure-ments were performed before (day 0), during (days 1, 3 and 7) and after (day 14) the chilling period

The net CO2assimilation rate was measured using a LI-6400 infrared gas exchange analyser (LI-COR Inc Lincoln, NE, USA) CO2assimilation was measured at

400 μmol m−2s−1constant light and 380 ppm constant external CO2 concentration, around the midpoint of leaf 4 blade Measurements were performed at least 4 h after light-on

Pigments and sugars analyses 8-mm leaf discs were harvested from leaf 4 blade,

15 cm from the ligule, on either side of the midrib Sampling was performed 5–7 h after light-on (between 2:00 and 4:00 p.m.)

For pigment extraction, one leaf disc was ground in li-quid nitrogen and the powder was extracted twice with

1 ml of methanol (once overnight at −80 °C, once for

30 min at−20 °C) Pooled supernatants were filtered on 0.45 μm syringe filters 100 μl were used for high-performance liquid chromatography using a Shimadzu

set-up (Prominence series, Shimadzu, Kyoto, Japan)

comprising a pump (LC-20AT), an autosampler

(SIL-20 AC) and a photodiode array detector (SPD-M(SIL-20A) A

Nova Pak C18 column (3.9 × 150 mm, 4 μm pore size)

from Waters (Ireland) was used for separation

Acquisi-tion and data analysis were performed using the

Em-power software (Shimadzu) Separation was obtained

with the following program: 2-min gradient from 100 %

solvent A (80 % methanol: 20 % 100 mM ammonium

acetate pH7) to 100 % solvent B (90 % acetonitrile in

water); 23-min gradient from 100 % B to 31 % B : 69 %

C (100 % ethylacetate); 10-min gradient from the latter

solvent mixture to 100 % A The solvent flow rate was

1 ml min−1 All solvents were HPLC grade and

ob-tained from VWR (Leuven, Belgium); commercial

pig-ments standards from DHI-Water and Environment

(Horstholm, Denmark) were used for calibration

De-termination of geranylgeranyl-chlorophyll a peak was

made according to [32] Total carotenoids include

neoxanthin, lutein,β caroten and the 3 pigments of the

xanthophyll cycle: violaxanthin (V), antheraxanthin (A)

and zeaxanthin (Z) A de-epoxidation index,

represent-ing the amount of zeaxanthin formed by conversion of

violaxanthin via the intermediate antheraxanthin, was

calculated as (A + Z)/(V + A + Z)

For sugar extraction, one leaf disc was ground in liquid

nitrogen and the powder was suspended in 1 ml of 80 %

ethanol: 20 % 100 mM Hepes-KOH (pH 7.1), 10 mM

MgCl2 as in [33] After incubation at 80 °C for 45 min,

tubes were cooled down to room temperature and

cen-trifuged 10 min at 4 °C, 13000 g Pellet and supernatant

were used for quantification of starch and soluble sugars,

respectively For starch analysis, the pellet was washed

twice with 40 mM sodium acetate pH 4.5, then

resus-pended in 200μl of sterile water and autoclaved 2 times

for 20 min at 120 °C to solubilize starch After

homogenization, quantification of starch was performed

with an enzymatic kit from R-Biopharm/Roche (cat nr

10207748035) following supplier instructions For

sol-uble sugar analyses, the supernatant was evaporated in

speedvac and the dry residue was dissolved in 1 ml of

sterile water Quantification of glucose, fructose and

su-crose was performed with an enzymatic kit from

R-Biopharm/Roche (cat nr 10716260035) Absorbance was

determined at 340 nm with a Lambda 20 UV/VIS

spec-trophotometer (Perkin Elmer, Norwalk, CT)

Transcriptional analyses

Samples were collected from leaf 4 mature zone (8-mm

discs taken 15 cm from the ligule), leaf 5 division zone

(segment 0–1.5 cm from the base) and leaf 5 elongation

zone (segment 3–4 cm from the base) Leaf tissues were

harvested 5–7 h after light-on (between 2:00 and

4:00 p.m.) and immediately frozen in liquid nitrogen

Samples from 10–15 plants were pooled, ground in li-quid nitrogen and stored at−80 °C until use Total RNA was extracted from 100 mg of leaf tissue with 1 ml TriR-eagent (Ambion, Applied Biosystems) Two phenol/ chloroform extractions were performed with two succes-sive purifications with 3 M potassium acetate pH 5.2 and 3 M sodium acetate pH 5.2 RNA was precipitated with isopropanol overnight at−20 °C RNA quantity and quality were controlled by absorption measurements at

230 and 260 nm (BioSpec-Nano, Shimadzu) After DNase treatment of total RNA (1 U DNase μg-1) for one hour at 37 °C, first-strand cDNA was synthesized from 1 μg RNA, using MMLV reverse transcriptase and oligo(dT)15 according to the manufacturer’s instructions (Promega) Aliquots were used as templates for qPCR with gene-specific primers (Additional file 1: Table S1)

In each 20-μl reaction, 3 μl cDNA and 10 μM of primers were used Reactions were performed in triplicate using SYBR Green I (Eurogentec) in 96-well plates with an iCycler IQ5 (Bio-Rad) For normalization, a geNorm-PLUS analysis was previously performed with ten house-keeping genes chosen from the literature [34, 35] Five genes were selected as constitutive genes (geNorm M value <0.5, see Additional file 2: Figure S1): CULLIN (CUL), UBIQUITIN (UBI), UBIQUITIN CONJUGAT ING ENZYME (UCE), FOLYLPOLYGLUTAMATE SYNTHASE (FGP1) and LEUNIG (LUG) The amplifi-cation efficiencies of all primer pairs were between 80 and 117 % (see Additional file 1: Table S1) The Rela-tive Expression Software Tool (REST, version 2009; http://rest.gene-quantification.info/), which operates

on Cq values, includes different PCR efficiencies and uses multireference genes for normalization [36, 37], was used for the relative quantification of qPCR data Statistical analyses

Data were analysed with R software (http://www.R-project.org/) We performed Student’s T tests for re-sults obtained from one single experiment; for repli-cate experiments, we pooled results when possible and performed analysis of variance in combination with means comparison (Tukey method) to distinguish sta-tistically different groups (P < 0.05) We fitted our data with linear mixed-effects models, using the lme4 package (http://CRAN.R-project.org/package=lme4), to take into account the effect of independent experiments (ran-dom effect)

Results Leaf growth is suspended in cold The growth of 6 inbreds cultivated in phytotronic cabi-nets at 24 °C day/18 °C night was recorded by regular measurement of the decimal visible leaf (VL) stage (Fig 1) At the 5-VL stage, one half of the plants were

exposed to a chilling-treatment of one week at 10 °C day/

7 °C night We extracted from Fig 1 the mean

phyllo-chron (time between successive leaf appearance) of

in-breds in control conditions, during (between day 0 and 7)

and after (from day 7 to 10-VL stage) chilling (Table 1)

During chilling, growth was almost arrested Among the 6

inbreds, the Flint line F03802, as well as the 2 Dent lines

B73 and Mo17 showed a complete stop, since the VL stage

after the low temperature treatment was not different than

before By contrast, the 3 inbreds F2, F283, and F353

kept on growing very slowly at 10 °C/7 °C After return

to warm conditions, growth recovered in all genotypes

up to control rate level (Table 1), so that the impact of

the treatment finally appeared as a short delay in VL

An end-point measurement of the final length of leaf 5

was performed at the 12-VL stage: in all genotypes, leaf

5 blade was 55-cm to 70-cm long in control conditions and was 10 % to 18 % shorter in plants that had been chilled for one week (Table 1) This negative effect of cold on leaf length was not visible on shoot biomass measured à 12-VL stage (Table 1)

The F2 and F03802 contrasted Flint lines were chosen for further analyses in hybrid context The F353 Dent line was chosen as the male tester, as it originates mainly from Iodent germplasm, a group well known for its good combining ability with Flint material [5] Since we ex-pected a higher number of leaves in hybrids as com-pared with the Flint parents and an heterotic effect

on plant tolerance to low temperatures, chilling was applied at a later stage (6-VL) and in harder condi-tions (10 °C day/4 °C night) than in the inbred exper-iments We observed that the rate of leaf emergence

4 6 8 10

4 6 8 10

Time [days from start of chilling treatment]

control chilling

Fig 1 Effect of chilling on leaf appearance rate in 6 inbred lines of maize The 7-day chilling treatment (10 °C day/7 °C night) was applied at the 5-VL stage (grey zone) Decimal leaf stage was measured on 15 plants per genotype; results shown are means ± sd Open symbols show plants grown in control conditions; filled symbols show chilled plants Upper line: Flint inbreds; lower line: Dent inbreds * indicates significant growth during chilling (slope different from 0)

Table 1 Effect of chilling treatment on leaf appearance rate, final length of leaf 5 blade and shoot biomass in 6 inbred lines of maize

The 7-day chilling treatment (10 °C day/7 °C night) was applied at 5-VL stage Measurements of final leaf length and biomass were performed at 12-VL stage Data

was similar in the hybrids than in the inbreds in

con-trol conditions, and that chilling caused a strong

in-hibition of growth (Fig 2a) Plants of hybrid

F03802xF353 completely stopped growing during the

treatment whereas F2xF353 continued very slowly

Phyllochron rose to normal values upon return to

warm conditions Thus, the hybrids behaved as their

Flint parental line F03802 and F2, respectively

In the experiments reported above, the effect of cold

was evaluated on the emergence of leaves, which occurs

during their linear phase of elongation This was

con-firmed by elongation rate measurements in the hybrid

experiment performed at 6-VL stage (Fig 2b) During

cold treatment, elongation of leaf 6 immediately ceased

in hybrid F03802xF353 but persisted at a very low rate

in F2xF353, for about 4 days In both hybrids, the

elong-ation rate returned to its initial value upon return to

warm temperature, but for a shorter time than the

dur-ation from appearance to the end of linear phase in

control conditions Consequently, the size of leaf 6 was significantly lower in cold-treated plants (Fig 2c) The blade was relatively more affected than the sheath (Fig 2d) The same measurements were per-formed on leaf 5, which was at the end of the linear phase of elongation at the start of the cold treatment, and on leaf 4 which had already entered the deceler-ating phase of leaf growth, i.e., had reached its final blade length Cold caused a severe reduction of elongation in the two leaves but, again, F2xF353 kept

on growing very slowly during the first days at low temperature whereas elongation completely stopped

in F03802xF353 By contrast, recovery was better in the latter hybrid Finally, leaf 5 was significantly shorter in cold-treated plants and the blade and sheath lengths were equally reduced by about 10 % (Fig 2c and d) Only the sheath of leaf 4 was reduced after cold, but there was no consequential and signifi-cant impact on total leaf length These effects of cold

a

c

d

b

Fig 2 Effect of chilling on leaf growth in two maize hybrids The 7-day chilling treatment (10 °C day/4 °C night) was applied at about the 6-VL stage (grey zone in a and b) a Leaf appearance rate Decimal leaf stage was measured on 15 –20 plants per genotype in each treatment; results shown are means ± sd for 7 experiments * indicates significant growth during chilling period (slope different from 0) b Leaf elongation rate of leaves 4, 5 and 6 Data are means ± sd of 15 plants for one experiment c Final length of leaves 4, 5 and 6 Data are means ± sd of 90 –106 plants (10–20 individuals in 6 experiments) * indicates significant effect of chilling (P < 0.05) d Leaf length deficit in chilling-treated plants, as % of controls

on leaf length were altogether insufficient to cause a

significant reduction in shoot biomass measured at

10-VL (data not shown)

Chilling effects on the photosynthetic machinery

We measured the maximum quantum efficiency of PSII

photochemistry (Fv/Fm), which is a good indicator of

PSII integrity [38] and the quantum yield of electron

transport in the light (ΦPSII) at 300 (inbreds) or 400

(hybrids) μmol m−2 s−1 PAR The use of excitation

en-ergy unaccounted for byΦPSII was estimated as in [31]

byΦNPQ, which is the fraction of light that is dissipated

thermally via regulated processes and by Φf,D, which is

the fraction of light that is lost by fluorescence (Φf) or

constitutive thermal dissipation (ΦD)

No significant difference was found between the three

parental inbreds for Fv/Fm measurements in control

conditions but more variability was found forΦPSII, F2

being the least efficient inbred (Fig 3) Chilling induced

a severe reduction of Fv/Fm from the first day for the

two Flint inbreds whereas F353 was significantly affected after one week of stress only For the three inbreds, ΦPSII was negatively impacted by chilling from the on-set, reflecting severe reduction of photochemical pro-cesses Amazingly, dissipation of the excess light energy did not occur by regulated heat emission but by fluores-cence or constitutive heat emission in the Flint inbreds F2 and F03802, as indicated by the decrease in ΦNPQ and the increase in Φf,D during chilling The situation was different in the Dent inbred F353 where an increase

in ΦNPQ, and hence in regulated thermal dissipation, was observed at the beginning of the chilling period and followed by an increase in Φf,D after 7 days of stress, when Fv/Fm was lower It is then noteworthy that varia-tions in Φf,D and Fv/Fm occurred in opposite patterns

in all three inbreds Seven days after the end of chilling (day 14, Fig 3), all parameters had recovered to control levels for all inbreds, indicating that the damages caused

by the treatment on the integrity and functioning of photosystems were reversible For Dent inbred F353,

Fig 3 Effect of chilling on chlorophyll fluorescence parameters in 3 inbred lines of maize The 7-day chilling treatment (10 °C day/7 °C night) was applied at about the 5-VL stage (grey zone) From the top to the bottom: maximal quantum yield of PSII (Fv/Fm); quantum yield of PSII in the light (300 μmol m −2 s−1PAR, ΦPSII); regulated thermal energy dissipation (ΦNPQ); constitutive thermal and radiative (fluorescence) energy dissipation ( Φf,D) Measurements were performed in the middle of 4th leaf blade before (day 0), during (days 1, 3 and 7) and after (day 14) the chilling treatment Data are means ± sd of 5 plants for one experiment * indicates significant effect of chilling (P < 0.05)

ΦPSII of treated plant was even higher after chilling

than that of control plants This observation suggests a

compensatory activation of photosynthesis after cold

but might also be explained by a decline of

photosyn-thesis efficiency in control conditions possibly due to

faster leaf senescence

The same analysis was performed with the two hybrids

F2xF353 and F03802xF353 As observed with the

par-ents, low temperature had a negative effect on Fv/Fm

and ΦPSII (Fig 4) Interestingly though, the impact on

Fv/Fm was significant after 3 days of chiling only, i.e.,

later than what was observed for the Flint parents F2

and F03802 (Fig 3), suggesting a benefit from the F353

Dent parent By contrast, ΦPSII had already decreased

after 1 day of chilling, indicating a reduction in electron

transport, and continued to do so until the end of the

treatment Most interestingly, dissipation of the excess

energy occurred by regulated and non-regulated

(consti-tutive) processes since both ΦNPQ and Φf,D increased

during the chilling treatment This result indicates that

the hybrids combined the dissipation strategies of their Flint and Dent parents shown in Fig 3

Chlorophyll and carotenoid contents of leaf 4 were compared in treated and control plants (Table 2) Tis-sues were harvested at the end of the chilling period for the treated plants, or 1 day after the beginning of the treatment for the control plants to compare plants

at the same VL stage In control conditions, F03802xF353 contained more chlorophyll (a + b) per unit leaf area than F2xF353 Chilling caused a strong decrease in chlorophyll content in both hybrids, but the chlorophyll a/b ratio showed little variation By contrast, there was a noticeable accumulation of the precursor geranylgeranyl chlorophylla, indicating a re-duction in chlorophyll a biosynthesis; this effect was stronger in F2xF353 than in F03802xF353 The carot-enoid content of the leaf was not very different in chill-ing versus standard conditions but whereas the xanthophyll pool was almost fully epoxidized in control conditions, the de-epoxidation index (de-epoxidation of

Fig 4 Effect of chilling on chlorophyll fluorescence parameters in two maize hybrids The 7-day chilling treatment (10 °C day/4 °C night) was applied at about the 6-VL stage (grey zone) From top to bottom: maximal quantum yield of PSII (Fv/Fm); quantum yield of PSII in the light (400 μmol m −2 s−1PAR, ΦPSII); regulated thermal energy dissipation (ΦNPQ); constitutive thermal and radiative (fluorescence) energy dissipation ( Φf,D) Measurements were performed in the middle of 4th leaf blade before (day 0), during (days 1, 3 and 7) and after (day 14) the chilling treatment Data are means ± sd of 29 plants (5 –8 individuals in 4 experiments) * indicates significant effect of chilling (P < 0.05)

violaxanthin via antheraxanthin to zeaxanthin)

in-creased to about 20 % in the cold, indicating the

activa-tion of the xanthophyll cycle The index was slightly

higher in F03802xF353 than in F2xF353

With regard to the activity of the C4 cycle, CO2

as-similation of leaf 4 in the light was negatively

im-pacted by cold treatment but respiration in the dark

was not (Fig 5a, Additional file 3: Figure S2) Starch and soluble sugars accumulated in the leaf of cold-treated plants (Fig 5b) This was particularly marked

in F03802xF353 hybrid that accumulated about 10 times more starch, sucrose and glucose in chilling conditions than in controls whereas the amplitude of variation was between 2 and 4 in F2xF353 In both

Table 2 Effect of chilling treatment on leaf pigment content in two maize hybrids

de-epoxidation index

[A+Z/V+A+Z, %]

chl chlorophyll, GG geranylgeranyl, A antheraxantin, Z zeaxanthin, V violaxanthin

The 7-day chilling treatment (10 °C day/4 °C night) was applied at about the 6-VL stage Sampling was made on leaf 4 blade (15 cm from the ligule, on either side of the midrib) at the end of the chilling treatment for treated plants or 1 day after the beginning of the treatment for control plants in order to compare plants at the same developmental stage Data are means ± sd of 21 plants (7 individuals in 3 experiments) Different letters indicate significant differences between groups (P < 0.05)

Fig 5 Effect of chilling on CO 2 assimilation, leaf sugar content and SLA in two maize hybrids The 7-day chilling treatment (10 °C day/4 °C night) was applied at about the 6-VL stage Analyses were performed on the 4 th leaf blade at the end of the chilling treatment for treated plants or

1 day after the beginning of the treatment for control plants in order to compare plants at the same developmental stage a CO 2 assimilation measured in the light (400 μmol m −2 s−1PAR) or in the dark at 25 °C, 380 μmol CO 2 mol−1 Data are means ± sd of 15 –20 plants (5–10 individuals

in 2 experiments) b SLA, soluble sugars and starch quantification Pie chart area is proportional to sugar amount (white disc in the center =

1 μg cm −2 scale) Different colours represent glucose (pink), fructose (green), sucrose (blue) and starch (purple) Data are means ± sd of 24 plants (8 individuals in 3 experiments) For SLA determination, data are means ± sd of 55 –60 plants (15–20 individuals in 3 experiments)

hybrids, SLA was lower after cold than in control

conditions (Fig 5b), which is consistent with the

ac-cumulation of carbohydrates

Effects of low temperature on gene expression

In order to see whether the effects of cold on leaf

elong-ation and photosynthesis could be correlated with

changes in gene expression, a panel of candidate genes

was selected from the literature and analysed by

RT-qPCR Two samples were taken in the growing zone of

leaf 5: one in the meristematic zone and one in the

elongation zone (Fig 6a) For photosynthetic

metabol-ism, leaf discs were harvested in the blade of leaf 4

where the Fv/Fm, ΦPSII, CO2 assimilation, pigments

and sugar analyses had been performed In all zones, the

transcript levels of DREB1 and ICE1 homologs were

quantified as markers of cold signalling [16, 39] and five

different constitutive genes were used for relative

RT-qPCR quantifications We found that ZmDREB1 was

induced at the end of the chilling treatment in the cell

division and cell elongation zones of leaf 5, whereas the

transcript levels of ICE1 were similar than in untreated

leaves (Fig 6, Additional file 4: Figure S3) The

up-regulation of ZmDREB1 was two times higher in

F03802xF353 than in F2xF353 Interestingly, the

opposite was observed in the mature zone of leaf 4, where ICE1 was strongly induced in low temperature conditions but not ZmDREB1 These results suggest that chilling signalling is different in growing non-photosynthetic tissues and in non-photosynthetic cells The transcripts of three cell-cycle genes were quanti-fied in the proliferation zone of leaf 5:CDKA1 (CYCLIN DEPENDENT KINASE A 1), CYCA3 (CYCLIN A 3) and KRP1 (CYCLIN-DEPENDENT KINASE INHIBITOR 1) were chosen after Rymen et al [15] showed that these genes were differentially expressed in the meristematic zone of leaves exposed to cold nights This was obvi-ously not the case in our experimental design since the relative transcript levels of the three genes were hardly different in chilled and control leaves In the elongation zone, two EXPANSIN genes, EXPA4 and EXPB2, se-lected from [40] were up-regulated in the cold In the mature zone of leaf 4, the list of candidates genes in-cluded CAB1, encoding a CHLOROPHYLL A/B BIND-ING PROTEIN of PSII [22],psbS and VIOLAXANTHIN DE-EPOXIDASE (VDE) both involved in energy dissipa-tion (ΦNPQ) (reviewed in [27]), the gene encoding GER-ANYLGERANYL REDUCTASE (GGR) that catalyses the terminal hydrogenation of geranylgeranyl chlorophyll a

to form chlorophylla [41] and genes encoding C4- and

Fig 6 Effect of chilling on gene expression in the leaves of two maize hybrids The 7-day chilling treatment (10 °C day/4 °C night) was applied at about the 6-VL stage Analyses were performed at the end of the chilling treatment for treated plants or 1 day after the beginning of the treatment for control plants in order to compare plants at the same developmental stage a Sampling procedure b Relative expression levels quantified by RT-qPCR Five genes were used for normalization Data are means ± se of 3 technical replicates for one experiment; two replicate experiments are shown in Additional file 4: Figure S3 Gene abbreviations: ICE1 (INDUCER OF CBF/DREB EXPRESSION 1), DREB1 (DROUGHT-RESPONSIVE ELEMENT BINDING), CDKA1 (CYCLIN DEPENDENT KINASE A 1), CYCA3 (CYCLIN A 3), KRP1 (CYCLIN-DEPENDENT KINASE INHIBITOR 1), EXPA4 (ALPHA EXPANSIN 4), EXPB2 (BETA EXPANSIN 2), GGR (GERANYLGERANYL REDUCTASE), CAB1 (CHLOROPHYLL A/B BINDING PROTEIN), psbS (CP22 PSII subunit), VDE (VIOLAXANTHIN DE-EPOXIDASE), PEPC (PHOSPHOENOLPYRUVATE CARBOXYLASE), PPDK (PYRUVATE, ORTHOPHOSPHATE DIKINASE) and rbcS (RUBISCO small subunit)